Project abstract Cardiovascular disease (CVD) remains the leading cause of death among older people. Age-associated mutations in blood cells of older people are newly discovered risk factors for CVD and hematologic malignancy (HM). The biological mechanisms underlying this risk are not yet known. Our study is limited to older women for the reasons we have described in the research plan but the research findings will have widespread application in older people. The most common acquired mutations occur in ASXL1, DNMT3A, and TET2, collectively referred as epigenetic regulatory genes (ERGs), result in loss-of-function and epigenetic dysregulation. Epigenetic mechanisms, e.g., DNA methylation and histone modification, regulate how monocytes function and respond to tissue injury. Pro-inflammatory monocytes can accelerate cholesterol-rich plaque formation in vessels and promote cancer development. Mutations that permanently change the epigenetic marks of monocytes in favor of inflammation are expected to produce detrimental consequences in human diseases. The overall goal of the project is to define the biological mechanisms that underlie the relationship between age-associated ERGs mutations in human monocytes and increased risk for CVD and HM. Fresh blood samples will be collected from older women enrolled in an ongoing prospective cohort study entitled Women Engaged in Advancing health Research (WEAR) conducted at Oregon Health & Science University. We will evaluate 50 older women with ASXL1, DNMT3A, or TET2 mutations (group 1), 50 older women without mutations matched for age and CV risk factors (group 2), and 20 younger women between 18-45 years old without mutations (group 3). Group 3 is included in this study to isolate the effect of aging. We have two specific aims to address the goals of the project: In aim 1, we will conduct genome-wide DNA methylation and RNA sequencing analyses and compare the data among the three study groups to define the pathogenic changes driven by ASXL1, DNMT3A, or TET2 mutations. The effects associated with aging will also be defined in this study. We hypothesize that age-associated mutations in ERGs induce a pathogenic DNA methylation and gene expression profile in monocytes characteristically associated with enhanced survival, proliferation, and inflammation. In aim 2, we will determine whether mutant ASXL1, DNMT3A, or TET2 monocytes (group 1) display phenotypic and functional abnormalities compared to control monocytes (groups 2 and 3) using established methodologies that evaluate monocyte activation and reactivity. We hypothesize that mutant monocytes display phenotypic and functional markers that are predictive of an exaggerated pro-inflammatory response to injury. The findings from these exploratory studies are expected to generate new hypotheses and support the design of experiments that precisely define how these age-associated, acquired genetic mutations in blood cells are manifesting as a disease syndrome involving multiple organs with poor health outcomes.